Improved viability and fertility of frozen-thawed dog sperm using adipose-derived mesenchymal stem cells
Ahmad Yar Qamar1,2, Xun Fang1, Min Jung Kim3
1Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, 34134, Republic of Korea.
Abstract:
Cryopreservation procedures negatively affect the quality traits of sperm, causing certain changes at structural and molecular levels due to thermal, mechanical, osmotic, and oxidative damage. The objective of this study was to examine the potential of canine adipose-derived mesenchymal stem cells (Ad-MSCs) for providing protection to the dog sperm against cryo-damage. Canine Ad-MSCs were selected on the basis of the significantly higher gene expression for different proteins actively involved in the cell repair including annexin 1 (ANX1), histone H3 (H3) and high mobility group B (HMGB) protein compared to skin fibroblasts. Semen was collected from four healthy dogs by digital manipulation. The washed pooled ejaculates were diluted with buffer 2 (extender) supplemented without Ad-MSCs (Control), with 2.5 × 106 Ad-MSCs/mL (Group 1) or with 5 × 106 Ad-MSCs/mL (Group 2). Group 1 exhibited significantly higher post-thaw motility, live sperm, intact plasma membrane and normal acrosomes than the other groups. Additionally, Group 1 showed significantly higher expression levels of genes related to the repair of membranes (ANX1, dysferlin; DYSF, and fibronectin; FN1) and chromatin material (H3 and HMGB). Protein expression of ANX1, H 3, and FN1 was also statistically more in Group 1 than in Control. The results confirm that canine Ad-MSCs can effectively preserve the quality of frozen-thawed sperm by a reduction in cryoinjury. At an appropriate concentration, Ad-MSCs significantly improve the quality of post-thaw dog sperm.
Insights
Canine adipose-derived mesenchymal stem cells (Ad-MSCs) protect dog sperm from cryopreservation damage. Supplementing semen with Ad-MSCs significantly improved post-thaw sperm quality and viability, reducing cryoinjury.
Area of Science:
- Reproductive Biology
- Stem Cell Therapy
- Cryobiology
Background:
- Cryopreservation of sperm causes structural and molecular damage, negatively impacting sperm quality.
- Canine adipose-derived mesenchymal stem cells (Ad-MSCs) possess cell-repairing properties, indicated by high gene expression of annexin 1 (ANX1), histone H3 (H3), and high mobility group B (HMGB) protein.
Purpose of the Study:
- To investigate the protective potential of canine Ad-MSCs against cryodamage in dog sperm.
- To evaluate the effect of Ad-MSCs on sperm quality parameters after cryopreservation.
Main Methods:
- Canine Ad-MSCs were selected based on superior gene expression for repair proteins compared to skin fibroblasts.
- Dog semen was cryopreserved with different concentrations of Ad-MSCs (Control, 2.5 × 10^6 Ad-MSCs/mL, 5 × 10^6 Ad-MSCs/mL).
- Post-thaw sperm quality, including motility, viability, plasma membrane integrity, and acrosome morphology, was assessed. Gene and protein expression related to repair mechanisms were analyzed.
Main Results:
- Semen supplemented with 2.5 × 10^6 Ad-MSCs/mL (Group 1) showed significantly higher post-thaw motility, live sperm count, plasma membrane integrity, and normal acrosomes compared to control and higher Ad-MSC concentration groups.
- Group 1 exhibited significantly increased gene expression for membrane repair (ANX1, DYSF, FN1) and chromatin material repair (H3, HMGB) genes.
- Protein expression of ANX1, H3, and FN1 was significantly higher in Group 1 compared to the control group.
Conclusions:
- Canine Ad-MSCs effectively preserve dog sperm quality during cryopreservation by mitigating cryoinjury.
- An appropriate concentration of Ad-MSCs significantly enhances the quality of post-thaw dog sperm, offering a promising therapeutic approach.
